Multidrug Idebenone/Naproxen Co‐loaded Aspasomes for Significant in vivo Anti‐inflammatory Activity

Abstract The use of proper nanocarriers for dermal and transdermal delivery of anti‐inflammatory drugs recently gained several attentions in the scientific community because they pass intact and accumulate payloads in the deepest layers of skin tissue. Ascorbyl palmitate‐based vesicles (aspasomes) can be considered a promising nanocarrier for dermal and transdermal delivery due to their skin whitening properties and suitable delivery of payloads through the skin. The aim of this study was the synthesis of multidrug Idebenone/naproxen co‐loaded aspasomes for the development of an effective anti‐inflammatory nanomedicine. Aspasomes had suitable physicochemical properties and were safe in vivo if topically applied on human healthy volunteers. Idebenone/naproxen co‐loaded aspasomes demonstrated an increased therapeutic efficacy of payloads compared to the commercially available Naprosyn® gel, with a rapid decrease of chemical‐induced erythema on human volunteers. These promising results strongly suggested a potential application of Idebenone/naproxen multidrug aspasomes for the development of an effective skin anti‐inflammatory therapy.

In vitro release kinetic profiles and percutaneous permeation studies. Release kinetic and percutaneous permeation studies were carried out by using Franz diffusion cells as previously reported with some modifications [4] . Synthetic cellulose (cut-off of 10,000 Da) and SCE membranes were used during the analysis [5] . All experiments with SCE membranes were performed in accordance with the Declaration of Helsinki, and the protocol was approved by the Research Ethics Committee of the University of Catanzaro "Magna Graecia" (Approval number: 390/2019). SCE membranes were obtained from the plastic reduction surgery of healthy adults (mean age 29 ± 4 years) and were isolated according to the published protocol [6] . The analysis was performed at 32.0 ± 0.5 °C. At fixed time points (30 min. and 1, 2, 3, 4, 5, 6, 8, 10, 12 and 24 hours), 500 μL of the receptor phase was withdrawn and replaced with fresh medium. The resulting samples were analyzed by UV-Vis spectrophotometer at 281 nm and 331 nm, for Idebenone and naproxen, respectively. An external calibration curve for both drugs was used during the analysis.
In vitro biosafety studies. Human keratinocytes cells (NCTC 2544) were used to evaluate in vitro biosafety of empty nanocarriers. The MTT test was used to investigate potential intrinsic cytotoxicity of nanocarriers. Cells were seeded in 96-well culture dishes (5 × 10 3 cells/0.2 mL) and after overnight incubation were treated with formulations 4A-son50% and 4B-son50% at the final lipid concentration of 100 µM. The cytotoxic effect of both samples was tested after 24, 48 and 72 hours of incubation. The formed formazan crystal was dissolved and the absorbance of the various samples was analyzed using an ELISA microplate reader (BIO RAD, xMarkTM Microplate Absorbance Spectrophotometer) at Abs 540 nm and 690 nm. The percentage of cell viability was calculated according to the following Equation (2): where, AbsT is the absorbance of treated cells and AbsC is the absorbance of untreated cells (negative control).
In vivo skin tolerability of empty aspasomes. Reflectance spectrophotometer was used on human healthy volunteers to evaluate biosafety of optimized aspasomes. This non-invasive technique is based on the use of reflectance spectrophotometer SP60 (X-Rite Incorporated, USA) for monitoring the erythema index (EI). For these studies, eight healthy human volunteers (mean age 26 ± 4 years) were enrolled after the signature of informed consensus, and the approval of experimental protocol. All details of the protocol were provided and explained before starting the study which was carried out in accordance with the Declaration of Helsinki, and the protocol was approved by the Research Ethics Committee of the University of Catanzaro "Magna Graecia" (Approval number: 392/2019). Healthy human volunteers were housed in the day surgery room 30 for min. before the experiments at room temperature (22 ± 2 °C and 40-50% room humidity). The analysis were provided as previously described with slight variations [6] . In vivo skin tolerability was evaluated after 24, 48 and 72 hours and was calculated according to the following Equation (3): where, 1/R is the inverse reflectance at a specific wavelength (510, 540, 560, 580, 610).
In vivo anti-inflammatory activity of multidrug aspasomes. Anti-inflammatory properties of Idebenone/naproxen co-loaded aspasomes were evaluated in vivo on eight healthy volunteers. Briefly, a chemical erythema was induced by methyl nicotinate solution (0.2% w/v) on specific skin area (1 cm 2 ). After the induction of cutaneous erythema, 200 µL of each formulation was applied to inflamed skin. At fixed time points (1, 2, 3, 4, 5, 6 hours), the variation of E.I. was evaluated. Saline solution (NaCl 0.9% w/v) and commercial 10% Naprosyn ® gel were used as negative and positive controls, respectively.
Statistical analysis. Statistical significance was evaluated by using One-way Anova test. A value of p < 0.05 was considered statistically significant. This analysis was performed with Microsoft Excel ® (Microsoft Corp., Redmond, WA) and GraphPad Prism. Figure S1. Variation of Backscattering (ΔBS%) and Transmission (ΔT%) profiles of aspasomes synthesized by using different lipid molar ratio (according to Table 1 included in the main text). Analysis was performed at 25 ± 0.5 °C and the resulting data are representative of three independent measurements.